Abstract
Purpose: :
To determine the effect of substratum compliance and Latrunculin B (Lat B) on Transforming Growth Factor beta-1 (TGF-β1)-induced differentiation of corneal fibroblasts to myofibroblasts. Corneal stromal haze is associated with myofibroblast appearance within corneal wounds. Substratum compliance modulates a variety of cellular behaviors as well as their response to therapeutic agents. Lat B is an actin cytoskeleton disruptor that is currently in clinical trials as a treatment for glaucoma but its effects on corneal cell subtypes have not been fully investigated.
Methods: :
Substrates with elastic moduli of 5 and 25 kPa stiffness as well as tissue culture polystyrene (TCP >1 GPa stiffness) were used in this study. The 5 kPa hydrogels approximate the modulus of normal rabbit stroma and 25 kPa hydrogels mimic the modulus of normal human stroma; TCP served as a control. Primary rabbit corneal fibroblasts were seeded in 6-well plates on substrates and cultured with media containing 0 or 1 ng/ml TGF-β1 for 72 hrs. After adhering to the surfaces for 24 h, cells were treated for 30 min with DMSO (control) or 0.4 μM Lat B in saline every 24 h for 72 h. RNA was harvested 4 h after DMSO or Lat B treatment. Quantitative PCR was performed to determine mRNA expression of myofibroblast phenotypic marker α-smooth muscle actin (α-SMA) and the predominant rabbit corneal crystalline aldehyde dehydrogenase 1A1 (ALDH1A1) which decreases with injury-induced stromal haze.
Results: :
In the presence or absence of TGF-β1, α-SMA mRNA expression was dramatically decreased on the 5 and 25 kPa hydrogels in comparison to TCP following DMSO treatment. Cells on the 5 and 25 kPa hydrogels showed further reductions in α-SMA mRNA following Lat B treatment regardless of TGF-β1 concentration. In contrast, cells grown on TCP and treated with Lat B produced a similar amount of α-SMA mRNA in comparison to DMSO treatment in the absence of TGF-β1. ALDH1A1 mRNA expression was increased on the 5 and 25 kPa gels following Lat B versus DMSO treatment at 1 ng/ml TGF-β1.
Conclusions: :
Substratum compliance modulates myofibroblast differentiation and corneal crystallin ALDH1A1 expression as well as expression of these proteins subsequent to Lat B treatment. The data also suggest that Lat B could have an effect on wound healing processes in the cornea through modulating stromal cell differentiation. Integrating the use of biologically relevant substratum compliance gives important insights into corneal cell reactions to drugs that may be more predictive of responses observed in vivo.
Keywords: cornea: stroma and keratocytes • cytoskeleton • wound healing